The recent development of genomic amplification with transcript sequencing (GAWTS) has greatly facilitated the sequencing of mutant alleles. Accordingly, over a five year period, the causative mutation in 200 unrelated individuals with hemophilia B (100 with severe, 60 with moderate, and 40 with mild disease) will be delineated by sequencing 11 critical regions (2.8 kb) of the factor IX gene. The sequence data will aid in the elucidation of the role of particular sequences in the expression, processing, secretion, or functioning of factor IX. Since X-linked lethal disease such as severe (and to some extend moderate) hemophilia B offer an opportunity to examine mutations that have occurred in the population within the last few generations, analysis of the sequence data will address several questions including: (1) Are there hypermutable or hypomutable sequences? In particular, is CpG a hotspot of mutation as recently suggested for hemophilia A? (2) How do the relative frequencies of mutational types vary with severity of disease? (3) Are there a few mutations which predominate in mild disease while virtually each family has its own mutation in severe disease? (4) Is there genetic heterogeneity, i.e., are there genes on the X chromosome which are necessary for the proper tissue expression or processing of factor IX? In families where the pedigree indicates that the origin of mutation might potentially be determined, GAWTS and DNA haplotype analysis will be performed on appropriate family members in order to estimate the sex ratio of mutation and rate of germ line mosaicism. An additional component of the project involves the development of methodology to further increase the power of GAWTS and to extend the technique to allow the rapid and very sensitive detection of factor IX mRNA, hopefully in readily detectable tissues such as blood, skin, or hair roots.